US7179795B2

US7179795B2 - Nucleic acids encoding chimeric proteins comprising BMP-2 and a proteinase inhibitor

Info

Publication number: US7179795B2
Application number: US10/257,384
Authority: US
Inventors: Bernd Wiederanders; Gunter Maubach
Original assignee: DePuy Biotech Jena GmbH
Current assignee: BIOTECH JENA GmbH; DePuy Biotech Jena GmbH
Priority date: 2000-04-18
Filing date: 2001-04-18
Publication date: 2007-02-20
Anticipated expiration: 2021-04-18
Also published as: AU7384801A; US20040087524A1; WO2001078756A2; NZ522038A; DE10020125A1; CA2406590A1; WO2001078756A3; AU2001273848B2; EP1276877A2; JP2004500124A

Abstract

This invention relates to an agent for producing a pharmaceutical drug for postoperative use after removal of bone tumors produced from a nucleic acid by linking a known sequence for promoting bone growth and a known proteinase inhibitor by a variable spacer molecule. This linkage results in a novel bifunctional active ingredient combining both properties in a biological molecule. This invention is used in the medical field, in particular in the specialty field of orthopedics.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 National Phase Entry Application from PCT/DE01/01510, filed Apr. 18, 2001 and designating the United States. This application further claims foreign priority to German application DE 100 20 125.3, filed Apr. 18, 2000.

This invention relates to an agent for postoperative use after removal of primary or metastatic bone tumors. This agent applies within the medical field, in particular in the field of orthopedics.

The prognosis for malignant bone tumors has undergone a definite change in the last two decades. In 1970 the five-year survival rate was less than 20%, but today almost 80% of patients survive. This success is attributed to recent therapeutic approaches with pre- or postoperative chemotherapy and/or radiation as well as expansion of the diagnostic and surgical options, which permit a differentiated surgical procedure according to entity, tumor extent and grading. The goal of surgical treatment, apart from a few exceptions, is complete removal of the tumor. There are various options for the procedure in removing a tumor:

(1) preserving the extremity in the original shape, bridging any resulting bone defect (limb salvage),
(2) segment amputation,
(3) amputation,
but method (1) is preferred in terms of the patient's quality of life. The following table shows the different resection limits with their pathological results:

Resection limits and pathological evaluation

	Resection level	Pathological result

Intracapsular	Intralesional	Resection margin in the
		tumor
Marginal	Extracapsular, but	Reactive tissue,
	in the accompanying	possibly with satellite
	reactive tissue	lesions of the tumor
Extensive	In normal tissue,	Tumor-free, possibly
	outside of reactive	dislocated metastases
	tissue (2 to 3 cm)	excised
Radical	Extracompartmental	Tumor-free resection
		margin

The risks entailed in surgery that salvages the extremity is apparent from the pathological findings, because individual tumor residues may remain if resection is inadequate. These tumor residues can have an extremely negative effect on the patient's prognosis (T. Ozaki, A. Hillmann, N. Lindner, S. Blasius, W. Winkelmann: Chondrosarcoma of the pelvis, Clin. Orthop. 337, 1997, 226–239). In the case of malignant sarcomas of bone and soft tissue, essentially extensive or radical resection is the goal (S. Toma, A. Venturino, G. Sogno, C. Formica, B. Bignotti, S. Bonassi, R. Palumbo: Metastatic bone tumors. Nonsurgical treatment. Outcome and survival, Clin. Orthop. 295, 1993, 246–251). Otherwise, a local recurrence rate of 60–90% must be assumed for marginal resections.

Pre- and/or postoperative treatment of bone tumors by chemotherapy or radiation therapy minimizes the recurrence problem. In addition to the known side effects of this treatment, however, renewed surgical procedures are repeatedly required in a certain percentage of these patients. In addition, not all tumors respond identically to the same treatment strategies. The high incidence of recurrences with a marginal resection in many cases makes an extensive or radical resection appear to be the preferred surgical method. However, the result of such surgery is usually that reconstruction of the bone proves to be complicated. Various options are available for reconstruction of bone:

- autologous reconstruction,
- endoprosthetics and
- allogenic implants.

Autologous reconstruction uses bone material from the patient, which is inserted in place of the bone removed. The possible removal of material is limited here, so that only minor resections can be refilled again. Endoprosthetics consist of replacing the missing bone by prostheses of biocompatible materials. This bone substitute is to some extent very complicated and cost-intensive to manufacture. In treatment of young patients in particular, the problem arises that the prostheses do not adapt to the patient's growth, thus necessitating follow-up surgeries. Allogenic implants presuppose the existence of a functioning bone bank. There is a high incidence of complications in allogenic bone and joint replacement in the traditional sense without a vascular connection. The fracture rate over a period of years is more than 50% in the case of diaphysis implants of the lower extremities, and the infection rate is reported as approximately 10% to 30%.

More recent therapeutic approaches are based on implantation of biodegradable materials coated with recombinant growth factors (C. A. Kirker-Head, T. N. Gerhart, S. H. Schelling, G. E. Hennig, E. Wang, M. E. Holtrop: Long-term healing of bone using recombinant human bone morphogenetic protein 2, Clin. Orthop. 318, 1995, 222–230). Preliminary experiments with recombinant growth factors have already been conducted on animal models (review article: E. H. Groeneveld and E. H. Burger: Bone morphogenetic proteins in human bone regeneration, Eur. J. Endocrinol. 142(1), 2000, 9–21). Methods of producing and using such recombinant growth factors are already known (U.S. Pat. No. 4,472,840, U.S. Pat. No. 4,563,489, U.S. Pat. No. 4,596,574, U.S. Pat. No. 4,789,732, U.S. Pat. No. 4,795,804, U.S. Pat. No. 5,318,898, U.S. Pat. No. 5,393,739, U.S. Pat. No. 5,618,924, European Patent 0,409,472, German Patent 19 748 734). These growth factors serve the purpose of improving the healing of bone defects because they stimulate natural bone growth. However, these factors have no effect on the persistence and possible dissemination of any tumor cells that may still be present postoperatively.

It is also known that the proliferation of osteoblasts may be increased and bone resorption of osteoclasts can be inhibited by a fragment of the HMW (high molecular weight) kininogen known as cysteine proteinase inhibitor (U.S. Pat. No. 5,885,964). The use of this fragment is appropriate when osteogenesis by osteoblasts is diminished due to age and bone resorption by osteoclasts is increased at the same time.

Furthermore, studies of the proteinases involved in tumor metastases and in bone resorption are known (C. Haeckel et al.: Proteinase expression in dedifferentiated parosteal osteosarcoma, Arch. Pathol. Lab. Med. 123, 1999, 213–221; K. Bjornland et al.: S1000A4 involvement in metastasis: deregulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in osteosarcoma cells transfected with an anti-S100A4 ribozyme, Cancer Res. 59, 1999, 4702–4708). The most important enzymes involved in this process are cysteine proteinases (cathepsins L, B), matrix metal proteinases (MMP-2, MMP-9) and the serine proteinase uPA. In addition, it is known that cathepsin K, a cysteine proteinase of osteoclasts, is involved in bone resorption (P. Garnero et al.: The collagenolytic activity of cathepsin K is unique among mammalian proteinases, J. Biol. Chem. 273, 1998, 32347–32352).

Growth factors of the TGF-β superfamily such as the BMPs (bone morphogenetic proteins) are capable of inducing osteoneogenesis. One example is BMP-2, which is described in the following articles: H. Itoh et al.: Experimental spinal fusion with use of recombinant human bone morphogenetic protein 2, Spine 24, 1999, 1402–1405; K. Yoshida et al.: Enhancement by recombinant human bone morphogenetic protein-2 of bone formation by means of porous hydroxyapatite in mandibular bone defects, J. Dent. Res. 78, 1999, 1505–1510. In addition, other BMPs have been described by J. M. Wozney et al.: Novel regulators of bone formation: molecular clones and activities, Science 242, 1988, 1528–1534; S. Oida et al.: Cloning and sequence of bone morphogenetic protein 4 (BMP-4) from a human placental cDNA library, DNA Seq. 5, 1995, 273–275; A. J. Celeste et al.: Identification of transforming growth factor-beta family members present in bone-inductive protein purified from bovine bone, Proc. Natl. Acad. Sci. USA 87, 1990, 9843–9847; E. Ozkaynak et al.: OP-1 cDNA encodes an osteogenic protein in the TGF-beta family, EMBO J. 9, 1990, 2085–2093; E. Ozkaynak et al.: Osteogenic protein-2, A new member of the transforming growth factor-beta superfamily expressed early in embryogenesis, J. Biol. Chem. 267, 1992, 25220–25227; J. Hino et al.: cDNA cloning and genomic structure of human bone morphogenetic protein-3B (BMP-3b), Biochem. Biophys. Res. Commun. 223, 1996, 304–310.

Endogenous proteinase inhibitors are also known. The sequence for human cystatin C was described by M. Abrahamson et al.: Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin, C. FEBS Lett. 216, 1987, 229–233; the sequence of TIMP-2 was described by W. G. Stetler-Stevenson et al.: Tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA expression in tumor cell lines and human tumor tissues, J. Biol. Chem. 265, 1990, 13933–13938 and that of PAI-2 was described by R. D. Ye et al.: cDNA cloning and expression in Escherichia coli of a plasminogen activator inhibitor from human placenta, J. Biol. Chem. 262, 1987, 3718–3725.

The object of this invention is to create an agent for postoperative use, i.e., after excision of primary or metastatic bone tumors, which supports successful bone regeneration and requires a less stressful surgery for the patient without the risk of a new tumor metastasis to the treated bone. The patient's quality of life should be increased through this type of bone resection with effective and long-lasting control of tumor, whereby it is necessary to take into account not only the surgical procedure for bone resection per se but also the consequences of the procedure.

According to this invention, an agent is prepared from a nucleic acid by linking an essentially known sequence for bone growth promotion and a known proteinase inhibitor by a variable spacer molecule, e.g., an oligonucleotide. This linkage results in a novel active ingredient having two functions.

When this bifunctional agent is used postoperatively after removal of bone tumors, bone growth is supported and also metastasis (through tumor cells remaining in the surgical field) in the marginal zones of the bone prosthesis is inhibited.

Depending on the biological activity of the tumor, micro-metastases may be expected in any case, leading to local relapses. In addition to the favorable effect on reconstitution of the bone, the risk of a possible metastasis should be largely minimized. In contrast to practice, it is to be done without a radical resection which has the goal of removing as much bone material that the resection borders are reliably tumor-free in order to effectively combat the tumor. Instead, only a minimum of bone material needs to be resected, whereby the risk of a further metastasis starting from the resection margin is reduced. Because the procedure is minimal, the patient's quality of life is increased not only concerning the surgery and its immediate consequences but also regarding later consequences. Due to the influence of the bifunctional agent, there is also better growth into the prosthesis, which results in shorter recovery times and more stable incorporation of the prosthesis, among other effects. This should also prevent follow-up surgeries.

A DNA according to the invention is described below in the form of a cDNA. This stands exemplarily forany DNA falling under the present invention. The agent is further described as a bifunctional protein and is produced with the help of well-known methods of genetic engineering. The basis of the bifunctional protein may be two independently naturally occurring proteins or domains of proteins, which are linked with one another by means of a spacer molecule (a peptide not belonging to the natural protein domains between the functional domains).

This invention includes the coupling of two cDNAs by an oligonucleotide to form a new cDNA. This invention also includes derivatives of this new cDNA, which are formed by replacement, insertion, or deletion of one or more nucleotides, where the activity of the coded gene product is preserved. An object of this invention is also recombinant expression of the bifunctional protein in prokaryotes such as E. coli strains. This is done by using vectors, which permit expression in prokaryotic cells. These vectors contain suitable well-known regulation signals for gene expression such as promoters and ribosome binding sites. The promotors used include, for example, the T7 promotor, the tac promotor and the tet promotor. The vectors also code for antibiotic resistence and the replication origin.

The cDNA of the bifunctional protein is used for in vitro and in vivo transfection of suitable cell cultures such as cells of mesenchymal origin. Transfection is understood to refer to the insertion of nucleic acid constructs into cells or tissue. To do so, vectors containing well-known and suitable regulation signals for gene expression are used. These include transcription signals such as promoters, enhancers, and polyadenylation sites as well as translation signals such as ribosome binding sites. Promotors used include eukaryotic promoters of viral and cellular origin such as the CMV promotor, the RSV promotor or the -actin promotor. All known polyadenylation signals may be used as the polyadenylation signal, e.g., that of SV40. These vectors may additionally contain genetic markers such as antibiotic resistance genes. Furthermore, viral vectors are also suitable for transfection of cells.

DNA constructs for prokaryotic and eukaryotic expression are produced by the well-known methods of genetic engineering such as PCR and cloning.

This invention will now be explained in greater detail on the basis of exemplary embodiments illustrated in the drawings, which show:


	FIG. 1	primer used in PCR
	FIG. 2	expression plasmid for the fusion protein of
		cystatin C and BMP-2
	FIG. 3	examples of possible oligonucleotides for linking
		the fusion partners
	FIG. 4	expression plasmid for the fusion protein of
		cystatin C and BMP-2 in a plasmid with
		maltose binding protein (MBP)

Embodiment 1:

Producing the Prokaryotic Expression Plasmid

The cDNA of human cystatin C was amplified by means of primers A and B shown in FIG. 1 for the polymerase chain reaction (PCR) from a plasmid (M. Abrahamson et al.: Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C, FEBS Lett. 216, 1987, 229–233). The cDNA of a mature human BMP-2 was obtained from the plasmid pBF008 (B. Fahnert, HKI Jena) by restriction digestion with the restriction endonucleases Eco47III and HindIII. Both cDNAs were cloned consecutively in reading frame in the expression plasmid pASK75 (Biometra). For this purpose firstly the PCR product of human cystatin C was cloned in the vector pCR2.1-TOPO (Invitrogen) and then the correctness of the PCR product was checked with the LICOR system. The cDNA of human cystatin C was cut out by restriction digestion with the enzymes XbaI and Eco47III and was ligated into the vector pASK75, which was cleaved with the same restriction enzymes. The resulting construct (pASKcC) was digested with the restriction enzymes Eco47III and HindIII. The cDNA of the mature human BMP-2 cut out of the plasmid pBF008 with the same restriction enzyme was ligated into the previously cleaved plasmid pASK75cC. A peptide consisting of six histidines (SEQ ID NO. 10), located on the N-terminus of the cDNA of mature human BMP-2, was used as a possible spacer between the two cDNAs in the cloning strategy described here (see also SEQ ID NOS. 12, 14, 16, 18). The base sequence in the regions of the DNA construct that were of interest was checked by DNA sequencing using the LICOR system, and it was verified that the reading frame is correct. FIG. 2 shows the resulting expression plasmid (pASKcCHBMP-2). FIG. 3 shows examples of possible oligonucleotides for linking the fusion partners. Exemplarily cystatin C is mentioned as an inhibitor and BMP-2 as a growth factor. For example, PAI-2 could also be used as a proteinase inhibitor for serine proteinases, or TIMP-2 could be used as an inhibitor for metalloproteinases. Other growth factors that could be used include BMP-3, BMP-4, BMP-7 and other representatives of the TGF-β superfamily.

The cDNA of the bifunctional protein (cCHBMP-2) was amplified by PCR with primers C, D and optionally D′ (FIG. 1) and inserted in the plasmid pMALc2 (New England BioLabs). For this purpose the PCR product cCHBMP-2 was cloned in the vector pCR2.1-TOPO and was cut out with the restriction enzymes BamHI and HindIII. The plasmid pMALc2 was digested with the same restriction enzymes. The two cDNAs were ligated together. The resulting expression plasmid was sequenced in the region of interest. This expression plasmid (pMALc2cCHBMP-2, FIG. 4) was used for expression of the bifunctional protein as a fusion protein with another protein, the protein MBP (maltose binding protein). MBP is used for purification of recombinant proteins by affinity chromatography. It can be removed again by factor Xa cleavage after purification, so that the authentic N-terminus of cystatin C is preserved.

Embodiment 2:

Expression of the Bifunctional Protein in E. coli (BL21(DE3))

For the purpose of expression, the expression plasmids pASKcCHBMP-2 (FIG. 2) and pMALc2cCHBMP-2 (FIG. 4), which are described in Embodiment 1, were transformed in the bacterial strain BL21 (DE3) (Novagen). The transformation was performed by well-known methods for chemically competent cells.

Expression was performed as follows. 200 mL Terrific broth (TB) medium with 100 g/mL ampicillin was inoculated with a single clone and cultured. Expression was induced by adding 100 g/L anhydrotetracycline in the case of plasmid paskcCHBMP-2 and by adding 1 mM IPTG in the case of plasmid pMalc2cCHBMP-2.

The bacteria were harvested by centrifugation and disrupted by well-known methods with lysis buffer and sonication. This material was purified by chromatography on Ni-NTA resin under denaturing conditions.

The folding and dimerization were performed in a dimerization buffer at 25° C. over a period of several days.

In addition to expression in bacteria, which is presented here as an example, expression in other known expression systems such as yeasts, insect cells or mammalian cells is also possible.

Claims

1. An isolated nucleic acid encoding a bifunctional protein comprising:

(a) an isolated nucleic acid encoding bone growth factor BMP-2 capable of inducing osteogenesis;

(b) an isolated nucleic acid encoding a proteinase inhibitor,

wherein said nucleic acid of (a) is linked to said nucleic acid of (b) via an oligonucleotide, and wherein said so linked nucleic acids encode a bifunctional protein.

2. The nucleic acid encoding a bifunctional protein of claim 1, wherein said nucleic acid encoding the proteinase inhibitor is a sequence encoding a cysteine proteinase inhibitor or a functional fragment thereof and wherein said functional fragment retains the function of said proteinase inhibitor encoded by said nucleic acid of (b).

3. The nucleic acid encoding a bifunctional protein of claim 1, wherein said nucleic acid encoding the proteinase inhibitor is a sequence encoding a serine proteinase inhibitor or a functional fragment thereof and wherein said functional fragment retains the function of said proteinase inhibitor encoded by said nucleic acid of (b).

4. The nucleic acid encoding a bifunctional protein of claim 1, wherein said nucleic acid encoding the proteinase inhibitor is a sequence encoding a metalloproteinase inhibitor or a functional fragment thereof and wherein said functional fragment retains the function of said proteinase inhibitor encoded by said nucleic acid of (b).

5. The nucleic acid encoding a bifunctional protein of claim 1, wherein the oligonucleotide linking (a) and (b) is SEQ ID NO: 10 or a functional fragment thereof.

6. The nucleic acid encoding a bifunctional protein of claim 1, wherein the oligonucleotide linking (a) and (b) is chosen from the group consisting of SEQ ID NO: 12, 14, 16, 18, or a functional fragment thereof.

7. A method for producing a bifunctional protein, said method comprising:

(a) providing an isolated nucleic acid encoding BMP-2 capable of inducing osteogenesis,

(b) linking said isolated nucleic acid to another isolated nucleic acid encoding a proteinase inhibitor via an oligonucleotide, and

expressing said bifunctional protein in vitro,

wherein said bifunctional protein retains the functions of said bone growth factor BMP-2 and said proteinase inhibitor.

8. The method of claim 7, wherein said nucleic acid encoding the proteinase inhibitor is a sequence encoding a cysteine proteinase inhibitor or a functional fragment thereof and wherein said functional fragment retains the function of said proteinase inhibitor encoded by said nucleic acid of (b).

9. The method of claim 7, wherein said nucleic acid encoding the proteinase inhibitor is a sequence encoding a serine proteinase inhibitor or a functional fragment thereof and wherein said functional fragment retains the function of said proteinase inhibitor encoded by said nucleic acid of (b).

10. The method of claim 7, wherein said nucleic acid encoding the proteinase inhibitor is a sequence encoding a metalloproteinase inhibitor or a functional fragment thereof and wherein said functional fragment retains the function of said proteinase inhibitor encoded by said nucleic acid of (b).

11. The method of claim 7, wherein the oligonucleotide linking the BMP-2 and proteinase inhibitor encoding sequences is SEQ ID NO: 10 or a functional fragment thereof.

12. The method of claim 7, wherein the oligonucleotide linking the BMP-2 and proteinase inhibitor encoding sequences is chosen from the group consisting of SEQ ID NO: 12, 14, 16, 18, or a functional fragment thereof.

13. A method for producing a nucleic acid encoding a bifunctional protein, said method comprising

(a) providing an isolated nucleic acid encoding a BMP-2 capable of inducing osteogenesis,

(b) linking said isolated nucleic acid to another isolated nucleic acid encoding a proteinase inhibitor via an oligonucleotide, wherein the so linked nucleic acids encode said bifunctional protein,

wherein said bifunctional protein retains the two functions of said BMP-2 and said proteinase inhibitor encoded by said nucleic acids of (a) and (b).

14. The method of claim 13, wherein the nucleic acid encoding the proteinase inhibitor is a sequence encoding a cysteine proteinase inhibitor or a functional fragment thereof.

15. The method of claim 13, wherein the nucleic acid encoding the proteinase inhibitor is a sequence encoding a serine proteinase inhibitor or a functional fragment thereof.

16. The method of claim 13, wherein the nucleic acid encoding the proteinase inhibitor is a sequence encoding a metalloproteinase inhibitor or a functional fragment thereof.

17. The method of claim 13, wherein the oligonucleotide is encoded by SEQ ID NO: 10 or a functional fragment thereof.

18. The method of claim 13, wherein the oligonucleotide is chosen from the group consisting of SEQ ID NO: 12, 14, 16, 18, or a functional fragment thereof.